Alison Mitchell

Cell biology: Explorers deliver tea to the pole

How does a fission yeast know where its ends are, so that the two poles can grow directly away from one another? New work indicates that a protein called tea1 acts as a marker, to direct the cellular growth machinery to the poles. Moreover, tea1 is localized to, and maintained at, the poles by the microtubular cytoskeleton.

In Vitro Reconstitution of the Late Steps of Genetic Recombination in E. coli

Purified proteins have been used to reconstitute an in vitro system for the medial-to-late stages of recombination in E. coli. In this system, RecA protein formed recombination intermediates that were processed by the actions of the RuvA, RuvB, and RuvC proteins. RuvAB was found to promote branch migration, to dissociate the RecA filament, and to modulate the orientation of cleavage of Holliday junction resolution by RuvC. Monoclonal antibodies directed against RuvA, RuvB, or RuvC inhibited resolution in the reconstituted system. Specific protein-protein interactions between the branch migration motor (RuvB) and the resolvase (RuvC) were also observed. These results provide evidence for coordinated action during the late stages of recombination, possibly involving the assembly of a RuvABC branch migration/resolution complex.

Role of RuvA in Branch Migration Reactions Catalyzed by the RuvA and RuvB Proteins of Escherichia coli

The RuvA and RuvB proteins of Escherichia coli promote ATP-dependent branch migration of Holliday junctions during homologous genetic recombination and DNA repair. In this process, RuvA acts as a specificity factor that targets RuvB, a hexameric ring motor protein, to the junction. Because elevated concentrations of RuvB can promote branch migration in the absence of RuvA, it has been suggested that RuvA acts as a molecular matchmaker. In the studies presented here, we compared the requirements for RuvAB- and RuvB-mediated branch migration reactions and found that reactions catalyzed by RuvB alone were highly sensitive to inhibition by NaCl, temperature, ADP, and ATPγS. Our observations indicate that the two reactions occur by distinct mechanisms and support the notion that RuvAB-mediated branch migration is physiologically more relevant than that catalyzed by RuvB. We also show that ongoing RuvAB-mediated branch migration reactions were blocked by the addition of polyclonal antibodies raised against RuvA. The role of RuvA is therefore unlikely to be restricted to RuvB targeting; instead, it is required continually during branch migration. Competition with excess synthetic Holliday junctions, sufficient to sequester released RuvA, failed to cause an immediate block and leads us to suggest that RuvAB promote branch migration by a processive mechanism.

The replication connection

The Mre11 complex — containing Mre11, Rad50 and Nbs1 — promotes the repair of double-strand DNA breaks (DSBs). One way to do this is by homologous recombination — a process that is intrinsically linked to DNA replication. Because of this connection, the Mre11 complex has been implicated in replication, and two new papers support this idea.

Signal transduction. A taste of things to come.

Supernova observations have indicated that the Universe is expanding faster than the theory of inflation predicts. Some theoretical cosmologists suggest that an exotic form of energy density called quintessence may be responsible. Quintessence began as Einsteins cosmological constant, but if it is not, in fact, constant it may explain why we appeared just when it had the same value as the density of ordinary matter.

A FLY'S EYE VIEW OF HUNTINGTON'S DISEASE

Animal models can often help in studying human diseases, and one such model is now described for Huntingtons disease. A characteristic of Huntingtons disease is long tracts of the amino acid glutamine in the huntington protein. These tracts have been engineered intoDrosophilaphotoreceptor cells, causing rapid neurodegeneration that mirrors the human condition.

UK women lead the way on interdisciplinary research

londonWomen scientists spend more of their time doing interdisciplinary research than their male counterparts, yet are less likely than men to be team players, according to the results of a survey due to be published next month.

A pushy protein: DNA recombination

NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 4 | APRIL 2003 | 261 In vitro biochemistry can tell us much, but the situation in vivo is often more complicated. Take homologous recombination, for example, which has been extensively studied using purified proteins and oligonucleotides. The Rad51 protein can catalyse pairing of homologous sequences and strand exchange in vitro, but what happens in chromosomes, where the DNA is wrapped around nucleosomes? Stephen Kowalczykowski and colleagues have addressed this question with their study of Saccharomyces cerevisiae Rad54, published in Nature Structural Biology. The Rad54 protein belongs to the SWI2/SNF2 group of ATPdependent chromatin-remodelling factors. These complexes allow DNA-binding factors access to the DNA by moving the nucleosomes out of the way. Rad54 has been shown to interact with Rad51 in vitro, where it stimulates Rad51-mediated strand exchange. This stimulatory effect could be explained if the function of Rad54 were to move nucleosomes out of Rad51’s path, so Kowalczykowski and colleagues first asked whether Rad54 can indeed redistribute nucleosomes on DNA. To study this, they reconstituted nucleosomes on short fragments of DNA, generating a mixture of nucleosomes at different places along the DNA. They then isolated three nucleosome species (N1, N2 and N3), which could be identified based on electrophoretic mobility — where the nucleosomes were closer to the centre of the DNA fragment (N3), the species migrated more slowly than if the nucleosomes were positioned nearer to the DNA ends (N1). Kowalczykowski and colleagues incubated the isolated nucleosomes with Rad54/ATP and, in each case, the nucleosomes became redistributed. The nucleosomes in N1 were moved to a more central position, whereas in N2 and N3 they were located closer to the DNA ends. Some free DNA was also generated, suggesting that some of the nucleosomes had been moved off the DNA fragments. The authors favour the idea that the nucleosomes were moved by sliding along — rather than by dissociating from, and then reassociating with — the DNA, as the amount of free DNA generated was greatest with the N1 species, where the nucleosomes had less far to travel to fall off the end. The authors next wondered where this chromatin-remodelling activity might fit in to the process of Rad51-mediated recombination. Rad51 forms a helical nucleoprotein filament on singlestranded (ss)DNA, which has previously been shown to stimulate Rad54’s other activities (ATPase and DNA-unwinding). So the authors examined the effects of incubating N3 and Rad54 with increasing amounts of Rad51/ssDNA. They found that the Rad51 complex enhanced the nucleosome-remodelling activity of Rad54 in a concentration-dependent manner. The optimal stoichiometry was one Rad54 monomer:one Rad51 monomer, suggesting that Rad54 might coassemble with the Rad51 nucleoprotein filament at an early stage of recombination — before the DNA-pairing and strand-exchange steps. The authors propose, then, that Rad54’s job in vivo could be to remodel chromatin and clear the DNA of nucleosomes while the recombinational repair machinery searches for homologous sequences. Interestingly, Rad54’s close association with Rad51 could also indicate a role for it after strand exchange, when it might clear Rad51 from the DNA to complete the repair process. Alison Mitchell References and links ORIGINAL RESEARCH PAPER Alexeev, A., Mazin, A. & Kowalczykowski, S. C. Rad54 protein possesses chromatinremodeling activity stimulated by the Rad51–ssDNA nucleoprotein filament. Nature Struct. Biol. 10, 182–186 (2003) FURTHER READING Tsukiyama, T. The in vivo functions of ATPdependent chromatin-remodelling factors. Nature Rev. Mol. Cell Biol. 3, 422–429 (2002) But more surprising still was the identification of PLCγ as a phosphorylation target for activated c-Abl in vivo. c-Abl complexes more tightly with PLCγ when it is active and through phosphorylation can inhibit PLCγ function, so forming an activation feedback loop. Although this activation mechanism will not be universal, it is the first link between c-Abl and phosphoinositide signalling, and has uncovered one new way in which to control activation of this tyrosine kinase. The work also shows how chemotaxis of cells towards a PDGF source requires active c-Abl.As c-Abl and PLCγ regulate the activity of one another, this work does not simplify the known roles of c-Abl,but further complicates the understanding of this kinase. Nothing in life ever seems easy, and c-Abl seems to need more than one mechanism to ensure it is ready for activation. Sarah Greaves, Senior Editor, Nature Cell Biology

DNA recombination: A pushy protein

NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 4 | APRIL 2003 | 261 In vitro biochemistry can tell us much, but the situation in vivo is often more complicated. Take homologous recombination, for example, which has been extensively studied using purified proteins and oligonucleotides. The Rad51 protein can catalyse pairing of homologous sequences and strand exchange in vitro, but what happens in chromosomes, where the DNA is wrapped around nucleosomes? Stephen Kowalczykowski and colleagues have addressed this question with their study of Saccharomyces cerevisiae Rad54, published in Nature Structural Biology. The Rad54 protein belongs to the SWI2/SNF2 group of ATPdependent chromatin-remodelling factors. These complexes allow DNA-binding factors access to the DNA by moving the nucleosomes out of the way. Rad54 has been shown to interact with Rad51 in vitro, where it stimulates Rad51-mediated strand exchange. This stimulatory effect could be explained if the function of Rad54 were to move nucleosomes out of Rad51’s path, so Kowalczykowski and colleagues first asked whether Rad54 can indeed redistribute nucleosomes on DNA. To study this, they reconstituted nucleosomes on short fragments of DNA, generating a mixture of nucleosomes at different places along the DNA. They then isolated three nucleosome species (N1, N2 and N3), which could be identified based on electrophoretic mobility — where the nucleosomes were closer to the centre of the DNA fragment (N3), the species migrated more slowly than if the nucleosomes were positioned nearer to the DNA ends (N1). Kowalczykowski and colleagues incubated the isolated nucleosomes with Rad54/ATP and, in each case, the nucleosomes became redistributed. The nucleosomes in N1 were moved to a more central position, whereas in N2 and N3 they were located closer to the DNA ends. Some free DNA was also generated, suggesting that some of the nucleosomes had been moved off the DNA fragments. The authors favour the idea that the nucleosomes were moved by sliding along — rather than by dissociating from, and then reassociating with — the DNA, as the amount of free DNA generated was greatest with the N1 species, where the nucleosomes had less far to travel to fall off the end. The authors next wondered where this chromatin-remodelling activity might fit in to the process of Rad51-mediated recombination. Rad51 forms a helical nucleoprotein filament on singlestranded (ss)DNA, which has previously been shown to stimulate Rad54’s other activities (ATPase and DNA-unwinding). So the authors examined the effects of incubating N3 and Rad54 with increasing amounts of Rad51/ssDNA. They found that the Rad51 complex enhanced the nucleosome-remodelling activity of Rad54 in a concentration-dependent manner. The optimal stoichiometry was one Rad54 monomer:one Rad51 monomer, suggesting that Rad54 might coassemble with the Rad51 nucleoprotein filament at an early stage of recombination — before the DNA-pairing and strand-exchange steps. The authors propose, then, that Rad54’s job in vivo could be to remodel chromatin and clear the DNA of nucleosomes while the recombinational repair machinery searches for homologous sequences. Interestingly, Rad54’s close association with Rad51 could also indicate a role for it after strand exchange, when it might clear Rad51 from the DNA to complete the repair process. Alison Mitchell References and links ORIGINAL RESEARCH PAPER Alexeev, A., Mazin, A. & Kowalczykowski, S. C. Rad54 protein possesses chromatinremodeling activity stimulated by the Rad51–ssDNA nucleoprotein filament. Nature Struct. Biol. 10, 182–186 (2003) FURTHER READING Tsukiyama, T. The in vivo functions of ATPdependent chromatin-remodelling factors. Nature Rev. Mol. Cell Biol. 3, 422–429 (2002) But more surprising still was the identification of PLCγ as a phosphorylation target for activated c-Abl in vivo. c-Abl complexes more tightly with PLCγ when it is active and through phosphorylation can inhibit PLCγ function, so forming an activation feedback loop. Although this activation mechanism will not be universal, it is the first link between c-Abl and phosphoinositide signalling, and has uncovered one new way in which to control activation of this tyrosine kinase. The work also shows how chemotaxis of cells towards a PDGF source requires active c-Abl.As c-Abl and PLCγ regulate the activity of one another, this work does not simplify the known roles of c-Abl,but further complicates the understanding of this kinase. Nothing in life ever seems easy, and c-Abl seems to need more than one mechanism to ensure it is ready for activation. Sarah Greaves, Senior Editor, Nature Cell Biology

Apoptosis: Death TRAIL

NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 3 | FEBRUARY 2002 | 81 The GTPase dynamin is crucial for vesicle fission during endocytosis and secretion. But can dynamin also influence actin dynamics? Initial evidence came from reports that dynamin interacts with actin-regulatory proteins and localizes at actin-rich sites. Two papers in the Proceedings of the National Academy of Sciences now show that dynamin can control actin nucleation from membranes, thereby regulating comet formation and movement. To explore a functional link between dynamin and actin, the groups studied the regulation of actin nucleation in actin comets. Comets are induced by infection with Listeria monocytogenes or by the accumulation of phosphatidylinositol 4,5-bisphosphate, which induces the activity of actin-regulatory proteins. The groups tagged dynamin 2 with green fluorescent protein (GFP) and stained for filamentous (F-)actin using phalloidin or the actin-binding protein cortactin. The pattern of GFP fluorescence strongly resembled that of F-actin in the comets. Dynamin was further enriched at the tips of vesicles near the membrane. Live imaging confirmed that dynamin is incorporated into the forming comets. But does it have an active function in these structures? To test this, both groups made use of dynamin mutants to assess any changes in actin tail formation or dynamics. GTPasedeficient dynamin–GFP mutants — which exert a dominantnegative effect on endocytosis — reduced the number and the speed of comets, and caused them to appear short and curled. The region of dynamin that directly binds to actin-regulatory proteins is its proline-rich domain (PRD). Its involvement in targeting dynamin to actin comets was studied using a PRDdeletion mutant (dynamin∆PRD–GFP), which led to fewer comets. Unlike the GTPase-deficient mutant, however, the comets were longer. Significantly, dynamin∆PRD–GFP couldn’t be detected in the comets, indicating that the PRD is required to target dynamin to these — and possibly other — actin structures. So, what is the functional role of dynamin in actin comets? Given its ability to bind to components of the actin-nucleating machinery, dynamin might regulate actin-nucleation. On the basis that it can also associate with the lipid bilayer, it could direct this nucleation to specific sites, such as the coated pits involved in endocytosis. It is also likely, however, that the interaction between dynamin and actin occurs at non-endocytic sites. Katrin Bussell References and links ORIGINAL RESEARCH PAPERS Lee, E. & De Camilli, P. Dynamin at actin tails. Proc. Natl Acad. Sci. USA 99, 161–166 (2002) | Orth, J. et al. The large GTPase dynamin regulates actin comet formation and movement in living cells. Proc. Natl Acad. Sci. USA 99, 167–172 (2002) The road to cell death involves two distinct routes — the ‘extrinsic’ and ‘intrinsic’ pathways, which proceed through death receptors or through mitochondrial events, respectively. Although these pathways converge at the level of effector caspases, they are thought to be completely separate before that. However, a report by Xiangwei Wu and colleagues in Genes and Development now adds to the growing evidence for crosstalk between these two pathways. They have discovered that death by an extrinsic pathway involving TRAIL relies on intrinsic, mitochondrial events to kill human cancer cells. Wu and co-workers used cells lacking Bax — a component of the intrinsic pathway — to show that this protein is needed for TRAIL-induced apoptosis. Moreover, whereas Bax was found in the cytosol before treatment with TRAIL, Bax translocated to the mitochondria after treatment.An inhibitor of the TRAIL-mediated extrinsic pathway prevented this translocation, suggesting that this movement depends on the extrinsic pathway. What’s the effect of Bax’s translocation to the mitochondria, and how does this link to the intrinsic signalling pathway? Wu and colleagues showed that the loss of Bax blocks the release of intrinsic signalling factors (namely cytochrome c and Smac/DIABLO) from the mitochondria. But whereas TRAIL-induced apoptosis could still occur without cytochrome c-mediated caspase activation, the liberation of Smac/DIABLO was crucial. Smac/DIABLO binds to (and removes the inhibitory effect of) a protein called XIAP, which normally inhibits caspase activity and blocks cell death. So Wu and colleagues propose that the TRAIL-mediated translocation of Bax allows the release of Smac/DIABLO from the mitochondria.This lifts the anti-apoptotic effects of XIAP,allowing cell death to proceed. Alison Mitchell References and links ORIGINAL RESEARCH PAPER Deng, Y., Lin, Y. & Wu, X. TRAIL-induced apoptosis requires Baxdependent mitochondrial release of Smac/DIABLO. Genes Dev. 16, 33–45 (2002) further defined, it offers an intriguing insight into how stem-cell renewal can be achieved and regulated. Simon Frantz